The primary goal of this proposal (Rapid and inexpensive epi/genetic profiling of the human mitochondrial genome) is to apply our new method of isolating and sequencing the mitochondrial genome (mtDNA) and epigenome (mtDNA-methylation) to large sets of samples in order to derive biological and clinical insights. We will characterize the variability in the mitochondrial genome and epigenome. The inexpensive and rapid method developed here will enable large-scale epidemiological studies of the role of mtDNA in aging and in the initiation and progression of a variety of human disorders. In specific aim 1 we will develop the techniques to efficiently sequence the mtDNA. We will sequence the mtDNA isolated from commonly used human cell-lines and human blood samples in order to establish a baseline for comparison with future studies. This will also help us understand the nature of variations supported by mtDNA. We will also develop analytical techniques to determine the quality of mtDNA isolation, assemble the reads into a complete genome and identify variations in the genome. In specific aim 2, we will modify and apply the technique of specific aim 1 to bisulfite treated mtDNA. This will allow us to profile DNA-methylation across the mtDNA in an unbiased manner. We will use the same samples as in specific aim 1 to quantify the levels of mtDNA methylation and its variability between samples, both within cell-lines and between individual blood samples. In specific aim 3 will apply the techniques of specific aims 1 and 2 to placental samples from a newborn cohort established at Mount Sinai School of Medicine. Since placentas are rich in mitochondria and hold the key to early development, we believe this will give us insights into developmental differences between newborns. We will correlate the phenotypes (birth weight, length and head circumference) to mtDNA profiles and generate hypotheses on the role of variants in determining the phenotypes. This will be the first epidemiological use of mtDNA profiling. The results of this proposal will have a broad impact on a range of fields, from bacteril genomics and genetics of chloroplasts in plants to disorders such as cancer and diabetes in humans.